Highlights d Open-source Python toolkit for 3D animal pose estimation, with DeepLabCut support d Enables camera calibration, filtering of trajectories, and visualization of tracked data d Tracking evaluation on calibration board, fly, mouse, and human datasets d Identifies a role for joint rotation in motor control of fly walking

Quantifying movement is critical for understanding animal behavior. Advances in computer vision now enable markerless tracking from 2D video, but most animals live and move in 3D. Here, we introduce Anipose, a Python toolkit for robust markerless 3D pose estimation. Anipose consists of four components: (1) a 3D calibration module, (2) filters to resolve 2D tracking errors, (3) a triangulation module that integrates temporal and spatial constraints, and (4) a pipeline to structure processing of large numbers of videos. We evaluate Anipose on four datasets: a moving calibration board, fruit flies walking on a treadmill, mice reaching for a pellet, and humans performing various actions. Because Anipose is built on popular 2D tracking methods (e.g., DeepLabCut), users can expand their existing experimental setups to incorporate robust 3D tracking. We hope this opensource software and accompanying tutorials (anipose.org) will facilitate the analysis of 3D animal behavior and the biology that underlies it.

All living things are profoundly affected by temperature. In spite of the thermodynamic constraints on biology, some animals have evolved to live and move in extremely cold environments. Here, we investigate behavioral mechanisms of cold tolerance in the snow fly (Chioneaspp.), a flightless crane fly that is active throughout the winter in boreal and alpine environments of the northern hemisphere. Using thermal imaging, we show that adult snow flies maintain the ability to walk down to an average body temperature of -7 °C. At this supercooling limit, ice crystallization occurs within the snow fly’s hemolymph and rapidly spreads throughout the body, resulting in death. However, we discovered that snow flies frequently survive freezing by rapidly amputating legs before ice crystallization can spread to their vital organs. Self-amputation of freezing limbs is a last-ditch tactic to prolong survival in frigid conditions that few animals can endure. Understanding the extreme physiology and behavior of snow insects is urgently important, as the alpine ecosystems they inhabit are being disproportionately altered by anthropogenic climate change.